We propose to continue our studies of the cell biology of hemopoietic stem cells. Recent progress in the molecular biology of colony-stimulating factors (CSFs) has not only made available most of the hemopoietic factors but has also revealed the presence of complex molecular and cellular mechanisms of hemopoiesis in culture. Therefore in order to elucidate the precise mechanisms of hemopoiesis, the development of a serum- free culture system for purified progenitors is necessary. We have partially succeeded in developing a serum-free culture system for enriched human progenitors based on modifications of alpha-medium. We propose to improve the technique and establish an optimal serum-free culture system for purified human hemopoietic progenitors. Our second aim is to continue to delineate the lineage specificities and targets of recombinant CSFs. Preliminary information on serum-free culture suggests that interleukin-3 (IL-3) is a stage-specific CSF and does not support the terminal process of hemopoietic differentiation. We recently discovered that B cell stimulatory factor (BSF-2) appears to possess synergistic activity with IL-3. We will characterize these factors in serum-free as well as serum-containing cultures of the purified hemopoietic progenitors. The third specific aim relates to the development of a primary clonal culture assay for B-lymphocyte progenitors. We have observed lymphocyte colonies containing mostly cells in the B cell lineage in high cell density culture of spleen cells obtained from 5-fluorouracil (5-FU)- treated mice in the presence of BSF-2. We will confirm the clonal nature of the colonies and develop a quantitative culture method for early lymphocyte progenitors. Finally, effects of recombinant CSFs will be tested in vivo in mice. We propose to test the hypothesis of stochastic stem cell differentiation in vivo and examine the mechanisms by which factors such as IL-1 alpha and BSF-2 interact with IL-3. The information to be obtained is likely to be pertinent to the understanding of the mechanisms of hemopoiesis in vivo and will provide basic information for the future use of CSFs in patients with hemopoietic disorders.